Project description:Analysis of a Bradyrhizobium japonicum pmtA mutant. PmtA catalyzes the first of three consecutive methylation reactions leading to phosphatidylcholine (PC) formation in B. japonicum. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. This study provides the first insight into global transcriptomic changes of a bacterial phosphatidylcholine biosynthesis mutant. Cells of the pmtA mutant and the wild type were grown to mid-exponential phase in full medium (PSY) under aerobic culture conditions. Keywords: genetic modification Comparative analyis of the B. japonicum pmtA mutant and the wild type grown under aerobic culture conditions.
Project description:Analysis of a Bradyrhizobium japonicum pmtA mutant. PmtA catalyzes the first of three consecutive methylation reactions leading to phosphatidylcholine (PC) formation in B. japonicum. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. This study provides the first insight into global transcriptomic changes of a bacterial phosphatidylcholine biosynthesis mutant. Cells of the pmtA mutant and the wild type were grown to mid-exponential phase in full medium (PSY) under aerobic culture conditions. Keywords: genetic modification Overall design: Comparative analyis of the B. japonicum pmtA mutant and the wild type grown under aerobic culture conditions.
Project description:Soybean plants host endosymbiotic dinitrogen (N2)-fixing bacteria from the genus Bradyrhizobium. Under oxygen-limiting conditions, Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum perform denitrification by sequentially reducing nitrate (NO3-) to nitrous oxide (N2O) or N2. The anaerobic reduction of NO3- to N2O was previously shown to be lower in B. japonicum than in B. diazoefficiens due to impaired periplasmic nitrate reductase (Nap) activity in B. japonicum. We herein demonstrated that impaired Nap activity in B. japonicum was due to low Nap protein levels, which may be related to a decline in the production of FixP and FixO proteins by the cbb3-type oxidase.
Project description:The recent identification of the iron response regulator (Irr) in Bradyrhizobium japonicum raised the question of whether the global regulator Fur is present in that organism. A fur gene homolog was isolated by the functional complementation of an Escherichia coli fur mutant. The B. japonicum Fur bound to a Fur box DNA element in vitro, and a fur mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus, B. japonicum expresses at least two regulators of iron metabolism.
Project description:When soil oxygen levels decrease, some bradyrhizobia use denitrification as an alternative form of respiration. Bradyrhizobium diazoefficiens (nos+) completely denitrifies nitrate (NO3-) to dinitrogen, whereas B. japonicum (nos-) is unable to reduce nitrous oxide to dinitrogen. We found that anaerobic growth with NO3- as the electron acceptor was significantly lower in B. japonicum than in B. diazoefficiens, and this was not explained by the absence of nos in B. japonicum. Our results indicate that the reason for the limited growth of B. japonicum is weak NO3- reduction due to impaired periplasmic nitrate reductase activity, which may rely on posttranscriptional events.
Project description:Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.
Project description:Iron utilization by bacteria in aerobic environments involves uptake as a ferric chelate from the environment, followed by reduction to the ferrous form. Ferric iron reduction is poorly understood in most bacterial species. Here, we identified Bradyrhizobium japonicum frcB (bll3557) as a gene adjacent to, and coregulated with, the pyoR gene (blr3555) encoding the outer membrane receptor for transport of a ferric pyoverdine. FrcB is a membrane-bound, diheme protein, characteristic of eukaryotic ferric reductases. Heme was essential for FrcB stability, as were conserved histidine residues in the protein that likely coordinate the heme moieties. Expression of the frcB gene in Escherichia coli conferred ferric reductase activity on those cells. Furthermore, reduced heme in purified FrcB was oxidized by ferric iron in vitro. B. japonicum cells showed inducible ferric reductase activity in iron-limited cells that was diminished in an frcB mutant. Steady-state levels of frcB mRNA were strongly induced under iron-limiting conditions, but transcript levels were low and unresponsive to iron in an irr mutant lacking the global iron response transcriptional regulator Irr. Thus, Irr positively controls the frcB gene. FrcB belongs to a family of previously uncharacterized proteins found in many proteobacteria and some cyanobacteria. This suggests that membrane-bound, heme-containing ferric reductase proteins are not confined to eukaryotes but may be common in bacteria.
Project description:The Bradyrhizobium japonicum ftsH gene was cloned by using a set of widely applicable degenerated oligonucleotides. Western blot experiments indicated that the FtsH protein was produced under standard growth conditions and that it was not heat inducible. Attempts to delete the ftsH gene in B. japonicum failed, suggesting a pivotal cellular function of this gene. The expression of B. japonicum ftsH in an ftsH-negative Escherichia coli strain significantly enhanced the fitness of this mutant and reduced the steady-state level of sigma(32).
Project description:The sdhCDAB operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont Bradyrhizobium japonicum. Sdh from B. japonicum is phylogenetically related to Sdh from mitochondria. This is the first example of a mitochondrion-like Sdh functionally expressed in Escherichia coli.
Project description:Bradyrhizobium elkanii is successfully used in the formulation of commercial inoculants and, together with B. japonicum, it fully supplies the plant nitrogen demands. Despite the similarity between B. japonicum and B. elkanii species, several works demonstrated genetic and physiological differences between them. In this work Representational Difference Analysis (RDA) was used for genomic comparison between B. elkanii SEMIA 587, a crop inoculant strain, and B. japonicum USDA 110, a reference strain. Two hundred sequences were obtained. From these, 46 sequences belonged exclusively to the genome of B. elkanii strain, and 154 showed similarity to sequences from B. japonicum genome. From the 46 sequences with no similarity to sequences from B. japonicum, 39 showed no similarity to sequences in public databases and seven showed similarity to sequences of genes coding for known proteins. These seven sequences were divided in three groups: similar to sequences from other Bradyrhizobium strains, similar to sequences from other nitrogen-fixing bacteria, and similar to sequences from non nitrogen-fixing bacteria. These new sequences could be used as DNA markers in order to investigate the rates of genetic material gain and loss in natural Bradyrhizobium strains.